Highlights A large proportion of analgesics fail to translate from rodents to humans. Large animals share anatomical and behavioral similarities to humans. Studying large animals can provide novel insights for arthritic pain. In vitro techniques developed in rodents can be adopted in large animals.

Objective Mesenchymal stem/stromal cells (MSCs) and MSC‐derived extracellular vesicles (MSC‐EVs) have been reported to alleviate pain in patients with knee osteoarthritis (OA). We undertook this study to determine whether MSCs and/or MSC‐EVs reduce OA pain through influencing sensory neuron excitability in OA joints. Methods We induced knee OA in adult male C57BL/6J mice through destabilization of the medial meniscus (DMM) surgery. Mice were sorted into 4 experimental groups with 9 mice per group as follows: unoperated sham, untreated DMM, DMM plus MSC treatment, and DMM plus MSC‐EV treatment. Treated mice received either MSCs at week 14 postsurgery or MSC‐EVs at weeks 12 and 14 postsurgery. Mouse behavior was evaluated by digging and rotarod tests and the Digital Ventilated Cage system. At week 16, mouse knee joints were harvested for histology, and dorsal root ganglion (DRG) neurons were isolated for electrophysiology. Furthermore, we induced hyperexcitability in DRG neurons in vitro using nerve growth factor (NGF) then treated these neurons with or without MSC‐EVs and evaluated neuron excitability. Results MSC‐ and MSC‐EV–treated DMM‐operated mice did not display pain‐related behavior changes (in locomotion, digging, and sleep) that occurred in untreated DMM‐operated mice. The absence of pain‐related behaviors in MSC‐ and MSC‐EV–treated mice was not the result of reduced joint damage but rather a lack of knee‐innervating sensory neuron hyperexcitability that was observed in untreated DMM‐operated mice. Furthermore, we found that NGF‐induced sensory neuron hyperexcitability is prevented by MSC‐EV treatment (P < 0.05 versus untreated NGF‐sensitized neurons when comparing action potential threshold). Conclusion MSCs and MSC‐EVs may reduce pain in OA by direct action on peripheral sensory neurons.

TitleFunctional characterization of ovine dorsal root ganglion neurons reveal peripheral sensitization after osteochondral defect.

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Objective: Knee joint trauma can cause an osteochondral defect (OD), a risk factor for osteoarthritis and cause of debilitating pain in patients. Modelling OD in rodents is difficult due to their smaller joint size. This study proposes sheep as a translationally relevant model to understand the neuronal basis of OD pain. Methods: Unilateral 6 mm deep OD was induced in adult sheep, 2-6 weeks after which dorsal root ganglion neurons (DRG neurons) were cultured from the control and OD side. Functional assessment of neuronal excitability and activity of the pain-related ion channels, TRPV1 and P2X3, was carried out using electrophysiology and Ca2+-imaging. Immunohistochemistry was utilized to verify expression of pain-related proteins. Results: An increased proportion of OD DRG neurons (sheep, n = 3, Ctrl neurons, n =15, OD neurons, n = 16) showed spontaneous electrical excitability (p = 0.009, unpaired t-test) and hyperexcitability upon TRPV1 agonist (capsaicin) application (p = 0.04, chi-sq test). Capsaicin also produced Ca2+ influx in an increased proportion of OD DRG neurons isolated (p = 0.001, chi-sq test). By contrast, neither protein expression, nor functionality of the P2X3 ion channel were altered in OD neurons. Conclusions: We provide evidence of increased excitability of DRG neurons (which is an important neural correlate of pain) and TRPV1 function in an OD sheep model. Our data show that functional assessment of sheep DRG neurons can provide important insights into the neural basis of OD pain and thus potentially prevent its progression into arthritic pain.

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Osteoarthritis (OA) is a common degenerative joint disease characterized by joint pain and stiffness. In humans, mesenchymal stem cells (MSCs) and derived extracellular vesicles (MSC-EVs) have been reported to alleviate pain in knee OA. Here, we used the destabilization of the medial meniscus (DMM) mouse model of OA to investigate mechanisms by which MSCs and MSC-EVs influence pain-related behavior. We found that MSC and MSC-EV treated DMM mice displayed improved OA pain-related behavior (i.e. locomotion, digging and sleep) compared to untreated DMM mice. Improved behavior was not the result of reduced joint damage, but rather knee-innervating sensory neurons from MSC and MSC-EV treated mice did not display the hyperexcitability observed in untreated DMM mice. Furthermore, we found that MSC-EVs normalize sensory neuron hyperexcitability induced by nerve growth factor in vitro. Our study suggests that MSCs and MSC-EVs may reduce pain in OA by direct action on peripheral sensory neurons.